JPS589687A - Production of polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field of the Invention The present invention relates to the field of recombinant DNA biotechnology. More specifically, the present invention relates to chymosin, which is an enzyme that coagulates milk, prochymosin, which is an enzyme precursor of chymosin and can be easily converted to chymosin.
It relates to a method for producing microorganisms capable of producing preprochymosin, the precursor of lc-prochymosin, from readily available materials.

TECHNICAL BACKGROUND OF THE INVENTION Milk essentially consists of a colloidal suspension of casein proteins. It has been known for more than 11 years that extracts of gastric juice in the abomasum of calves have a destabilizing effect on this colloid. The result of aggregation of casein molecules is a phenomenon commonly known as curdling.

This curd is the first step in cheese production.

During the first 2-3 weeks after a calf is born, the calf continues to drink milk from its mother while in the weaning cage. Digestion of this milk is aided by the presence of acid proteinases (collectively called rennins or rennets) in the calf's abomasum. Rennin has a proteolytic effect on catupacasein in milk, causing cleavage of the protein chains. The resulting protein (paracatupa casein) cannot be stabilized in colloidal solution and curdling occurs.

For many years, the only source of rennin for the cheese industry was calf stomachs. However, in recent years there has been a growth in the cheese manufacturing industry that has created a demand for rennin that exceeds the naturally available supply. Although breeding calves for slaughter within the first 12 weeks of birth is not economically viable for producing large amounts of natural rennin, it is easy to find alternatives. It wasn't a big deal.

Attempting to find natural alternatives to rennin requires acidic proteinases or mixtures of acidic proteinases with very low proteolytic activity. These conditions must be met in order to obtain a sufficient yield of curd and to ripen the cheese well; This causes the casein protein to split to the point where it breaks apart.

This is not a good thing. A particular disadvantage is that the smaller degraded components of casein protein impart a bitter taste to the final product, often associated with unpleasant odors in the minds of consumers.

Two curds have been accepted as alternatives in the cheese manufacturing industry.

One is derived from animal enzymes and consists of a mixture of bovine pepsin and calf rennin. (Bovine pepsin is an enzyme found in the stomach of adult cows and is a proteolytic enzyme that replaces rennin in unweaned calves.) Naturally occurring proteolytic enzymes of this composition are pure It is widely accepted as an alternative to calf rennin.

Second, and perhaps more importantly, proteolytic enzymes from fungi have been widely developed.

These enzymes of microbial origin were developed in Japan several years ago. The three main sources of emulsion curds of microbial origin are strains such as: These are Mucor psillaum, Mucorml.h@%, and Endothiaparasiticm.

A disadvantage of the emulsifying agents produced from such strains is that they generally have significant proteolytic activity. As mentioned above, this will reduce the production of curd and therefore the production of cheese. Bitterness may also occur. This kind of thing is
This is particularly noticeable in cheeses that require a long aging period.

For this reason, emulsifying agents of microbial origin are only practically applicable to cheeses that require short maturation.

The major proteolytic enzyme found in rennin is chymosin. This protein is produced from an inactive precursor called prochymosin, which is secreted from the calf's abomasum (abomasum). Chymosin has been an important research topic in recent years. One of the results of this research was the determination of the complete amino acid sequence of prochymosin, an enzyme precursor. (Foltman et al. Prec. Nat,
Acad, Sci. USA 772321-2334 (
1977) and J-Bloll. Chem. 2548447
See -8456 (1979). ) The first of these papers presents the complete 365 amino acid sequence of prochymosin. In the second paper, the sequence of a single peptide chain consisting of 323 amino acid residues, which is the main chain of chymosin, is described. Removal of 42 amino acids from the amino terminus of the prochymosin chain causes activation of the enzyme. We found that the major translation product of the calf stomach prochymosin gene is preprochymosin, a precursor with a 16 amino acid N-terminus to the prochymosin moiety. The present invention includes a method for producing calf chymosin, prochymosin, and preprochymosin from bacteria using recombinant DNA or genetic engineering techniques. Prochymosin is then autocatalytic, −
Can be divided by dependent reactions.

In accordance with the purpose of the present invention, we express methionine-chymosin, methionine-chymosin, and methionine-chymosin by a host organism transformed with a vector system carrying the coding gene for methionine-chymosin, methionine-prochymosin or preprochymosin, respectively. - Provides a method for producing chymosin comprising means for cleaving prochymosin or preprochymosin.

In one embodiment, we provide a method for producing chymosin comprising the following steps.

a) Insertion of the gene encoding methionine-prochymosin into the vector system.

b) Transformation of the host organism by the gene with vector system.

c) Cleavage of methionine-prochymosin expressed by the host organism to produce chymosin.

In another aspect of the invention, we provide a method for producing methionine-prochymosine comprising the following steps:

a) Insertion of the gene encoding methionine-prochymosin into the vector system.

b) Transformation of the host organism by the gene with vector system.

c) Isolation of methionine-prochymosin expressed by the transformed host organism.

In another aspect of the invention, we provide a method for producing preprochymosin comprising the following steps.

a) Insertion of the gene encoding preprochymosin into the vector system.

b) Transformation of the host organism by the gene with vector system.

c) Cultivation of the host organism for expression.

Preferably, in this embodiment, the preprochymosin expressed by the transformed host organism is isolated.

In another aspect of the present invention, we provide a method for producing methionine-chymosin comprising the following steps.

a) Insertion of the gene encoding methionine-chymosin into the vector system.

b) Transformation of the host organism by the gene with vector system.

c) Isolation of methionine-chymosin expressed by the transformed host organism.

In another aspect of the invention, we provide a method for producing prochymosin comprising the following steps:

a) Insertion of the gene encoding preprochymosin into the vector system.

b) Transformation of the host organism by the gene with vector system.

e) Cleavage of preprochymosin expressed by the transformed host organism to produce prochymosin.

In another aspect of the present invention, we provide a method for producing chymosin comprising the following means.

a) Insertion of the gene encoding preprochymosin into the vector system.

b) Transformation of the host organism by the gene with vector system.

c) Cleavage of preprochymosin expressed by the transformed host organism to produce chymosin.

In another aspect of the invention, we provide the polypeptides methionine-prochymosin, methionine-chymosin, and preprochymosin. Preferably, these are produced by the method of the embodiments of the invention described above. (We also provide chymosin produced by these methods.) In other aspects of the invention we provide genes encoding methionine-prochymosin, methionine-chymosin, and preprochymosin.

In another aspect of the invention, we provide a system comprising a gene encoding methionine-prochymosin, preprochymosin, or methionine-chymosin. The vector system can include a regulatable expression promoter sequence. In one preferred embodiment, the vector system is capable of transforming a bacterial host organism and imparting a phenotype to the transformed host organism that is different from an untransformed host organism. may contain one or more amino acid sequences capable of. Preferably, the vector system includes an E. coli tryptophan operon promoter. In a second, more desirable embodiment, the vector system is capable of transforming a eukaryotic host organism and imparts a phenotype to the transformed host organism that is different from an untransformed host organism. 4
can include one or more amino acid sequences that can be Preferably, the host organism is a yeast. More preferably, the vector system is derived from the bacterial plasmid pBR322 and the yeast 2μ plasmid. More preferably, the vector system contains a yeast phosphoglycerate kinase (PGK) gene promoter sequence.

In another aspect of the invention, we provide host organisms transformed by the vector systems described above. Preferably, the host organism is a bacterium, such as a suitable strain of E. coli such as HBIOI or RV308.

Alternatively, the host organism may be yeast, a suitable one being Satucharomyces cerevisiae (saeeha-rom
yees cerevisiae).

In other embodiments of the invention, we provide oligonucleotides comprising the following nucleotide sequences:

5'GTT, CAT, CAT, GTT3' where G is guanine, Td thymine, A is adenine and C is cytosine.

Preferably, this oligonucleotide is used for hybridization tests and in procedures for the detection of mRNA containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of chymosin. As a transcription glimer, a single-stranded cDNA encodes a polypeptide containing the amino acid sequence of chymosin.
Preferably it is used in an operation for preparing a portion of A.

In a final aspect of the invention, we provide a quantitative method for chymosin activity in which one or more dilutions of the sample are incubated with milk in the wells of a microtitration plate. Preferably, this quantitative method consists of: a) preparation of serial dilutions of the sample;

b) Addition of milk standard to each dilution.

c) Incubation for a given time.

d) Observation of whether the milk sample coagulates.

Preferably, the milk is dry skim milk that has been reconstituted into a liquid state.

DESCRIPTION OF THE EXAMPLES In order to fully explain the present invention, a general process will first be described, followed by details of the general process.

The first step in the production of microorganisms as required by the present invention is the isolation of the gene encoding the desired protein. In the case of prochymosin, the size of the protein and the redundancy in the genetic code make chemical synthesis of such a gene uninteresting. One research avenue for clonable genes is the preparation of prochymosin mRNA from calf gastric mucosa (Uchiyama et al.
ri*.

Blol, Chom441373-1381 (1980
)reference).

Isolation of mRNA can be performed using one of several methods, such as total RNA (Enxymlog
yVol) iAead meeting mi*Pr@ss (1968)
Kirby's method) or polysomal RNAQD extraction (Palmiter J, Biol, Ch@m, 248209
5-2106 (1973)).

/ liquimosin mRNA concentration, for example, oligo(dT)
It can be concentrated at an advanced stage of purification by cellulose column chromatography or sucrose density gradient centrifugation.

Once a fairly pure sample of prochymosin mRNA is made, this mRNA preparation can then be transcribed with AMV reverse transcriptase (Mtage et al.
AeldR・Res. μm221-1240 (1979
), thereby making single-stranded polychymosin eDNA
I can do it. This copy can be isolated and transcribed again by AMV reverse transcriptase or DNA polymerase I (Estrathiazis et al. Cell. 72
79-288 (1976)) produce a double-stranded full-length gene with a double-stranded gene connected at one end by a hairpin loop. This ruda is
Can be cleaved with S1 endonuclease (Bokuto Ear, J., Bioeh@m, 33192-200 (
(1973)) Since the use of *si nuclease results in the loss of some nucleotide sequence from the ends of the synthetic gene, an alternative method is used. in this way,
Using an enzyme called terminal transferase, a short segment of oligodeoxycytosine is transferred to the 3' strand of the first strand of eDNA.
(Nuel, Aeid)
Ram, 92251 (1981)), in which a short oligodeoxyguanosine glymer is added to the eDN of the second strand.
Used to proceed with A synthesis. In this procedure, the complete nucleotide sequence is available for cloning. A schematic diagram of the eDNA synthesis process is shown in FIG. (In this figure, the dashed line represents mRNA, and the solid line represents cDNA.
A).

The resulting full-length prochymosin gene is prepared for insertion into a suitable plasmid vector, for example by linking a synthetic binding agent to the prochymosin gene (Porter et al. Natur@2B2471-477 (1979).
)reference). This ligation can be performed using, for example, T4 DNA ligase. This binding agent is then cleaved using an appropriate restriction enzyme to create staggered binding sites for prochymosin DNAK. These binding sites are used for binding to plasmid vectors. or,
Prochymosin DNA can have short sites of oligodeoxycytosine attached to its ends and are hybridized to complementary sites of oligodeoxyguanosine attached to the ends of these plasmid vectors (Mafjilibray et al. (1980). ) Prow, Nat, Aead, Se
i.

USA775153-5157). Once a suitable vector is chosen, the intersecting cuts are placed into a ring structure using appropriate restriction enzymes, and the prochymosin DNA portion is ligated into the ring. The vector used initially is not capable of expressing the isolated gene. The vector chosen for expression must contain a strong promoter to express the inserted gene, and must also contain the same common gene that codes for resistance to antibodies and the like. The recombinant plasmid thus created is used to transform a suitable host organism (e.g. E. coli) (Cohen et al. (1972) Pro, Nat, Acad, Se 1.69.
2110-2114).

The prochymosin mRNA used in the initial steps to create this host organism is not completely pure;
Therefore, only some of the various eDNAs that are cloned have DNA sequences directly related to prochymosin. Therefore, a screening procedure is essential to identify which plasmids carry the prochymosin DNA sequence. This includes hybridization analysis using synthetic oligonucleotides with sequences partially specific to the amino acid sequence of prochymosin (Grunstein and Hogness (1975)).
ead, 8ei.

723961-3965 and 7 Ortman et al. (1977
) Proe.

Nat. Acad, 8ei, 742321-2334), using this procedure, prochymosin-specific clones can be detected from among the various clones created by the procedures up to this point. Full-length eDNAO copies can be selected from clones specific for prochymosin production by size fractionation using polyacrylamide gel electrophoresis, followed by digestion of plasmid DNA with restriction enzymes (Doul et al. 1980) Nuca1.
Aeid, Re1. 4575-4592), then
The selected plasmid is transferred to the expression vector of choice, which provides precise orientation and alignment of the insert with respect to plasmid control sequences. Once all complementary DNA genes in the correct configuration are obtained, gene expression can be achieved by standard processes of host cell metabolism and prochymosin production can still be determined using protein chemistry techniques known in the art. be measured. We have devised a procedure to isolate and purify prochymosin from microbial cells that involves subjecting the protein to acidic pH conditions. Prochymosin has acidic pH
It is known that chymosin can be converted to active chymosin in an autocatalytic process involving
4). This activation occurs from the amino terminus of polychymosin to
occurs by removing two amino acids (Peterson et al. (1979) Eur, J.

Biech@m, 94573-580).

A more detailed explanation of all the individual operations of the method will now be given.

In a specific example, preparation of RNA from calf stomach was performed as follows.

Slaughtered Bakar unweaned calf (age 10B)
The abomasum (abomasum) of each mouse was dissected and washed with isotonic buffer. The reddish mucosal layer was scraped off with a small blade and kept at 0°C. Tissues were used immediately in RNAp and frozen in liquid nitrogen prior to storage at -80°C. Approximately 50 gm of mucosa was obtained per stomach.

RNA is mainly obtained by Kirby's method (Essymolo
gy.

VolM, AeadsmlePress (1968) P
, 87) and isolated from the abomasum mucosa by the following method. The scraped abomasum mucosa was cut into small pieces with scissors before homogenization. 25 gm of tissue was treated with 45 ml of 1% sodium chloride (w/s), 45 ml of 6% sodium 4-aminosalicylate, 45 ml of phenol:cresol mixture (500 gm of phenol, 70 ml of m-cresol).
, water - 55 ml, 8-hydroxyquinoline - 0,5 gm
) mixed with The mixture was heated at 6000c:&, 30°C at 5°C.
Shake at room temperature for 20 minutes before centrifuging for minutes. The upper layer was removed and adjusted to 3% (v/v) with sodium chloride.

This layer was then extracted with this volume of a phenol: m-cresol mixture for 10 minutes at room temperature and then
Centrifuged for 15 minutes at 0xg and 50°C. excluding the aqueous layer,
It was mixed with twice the volume of ethanol and precooled at -20°C.

The mixture was kept at 0°C for 20 minutes to remove the precipitated RNA and DNA.
was centrifuged at 8000 xg and 5°C for 10 minutes. The nucleic acid pellet is 2% (w/v) in 100 ml at pH 6.0.
Gently resuspend in sodium acetate and the solution made to 3M with sodium chloride by addition of solids. The RNA precipitate was incubated at 8000 x g for 15 min at 5°C.
Centrifuged for minutes. The pellet contains DNA and soluble RNA.
Wash thoroughly with 3M sodium acetate, pH 6.0, and then wash with 70% (v/v) to remove sodium acetate.
v) Washed with ethanol. Then dry the pellets and
Redissolved in dilute buffer and stored at -20°C. What was obtained was approximately 30-40 mg of RNA.

This calf stomach RNA extract was further purified by the chromatography procedure described below. For purification, it may be possible to use, for example, the sucrose density gradient method.
20mM HEPES (pH 7.5), 2 EDTA, 0
．． In a buffer containing 1% SDS (loading buffer), 1 mg/
Prepare to ml. This solution was combined with 2 gm of oligo(dT)cellulose (B), which had been thoroughly washed with loading buffer.
, R,L.

Ltd., Bethesda, Md.). After loading the column under gravity, unbound RNA was washed away with loading buffer until the optical density of the extract was below 0.05 at 260 nm.

The bound mRNA portion was then treated with several column volumes of 20mM HEPEs (p) 17.5), 1mM EDTA.
, extracted with 0.1% BDB. mRNA at -20℃, 2
It was collected by precipitation with a volume of absolute ethanol, and the precipitate was centrifuged at 15,000 xg for 10 minutes at 4°C. 300-400 micrograms of mRNA portion was obtained.

The next step in the operation is to extract single-stranded e from prochymosin mRNA.
It is to prepare DNA. In order to create a full-length eDNA single strand from a single mRNA strand, it is necessary to grime the mRNA at a specific site in the mRNA where cDNA transcription can begin.

Two glimers were used in this work. One of them is oligo(dT) 12-18. This grima is
It is made up of a series of thymidine (T) residues.

A feature of almost all eukaryotic mRNAs is that they contain a series of adenine (A) residues at one end of the chain.

The above-mentioned glimer can intersect with this oligo(A) group and can serve as a starting point for the transcription of single-stranded eDNA. The following method is basically based on the method of Mtarget et al. (NueA, Ac1
d, R@1.61221-1240, (1979). The mixture contained 50mM Tris. Cl (pH 8,3), 1
0,5m labeled with 0mM magnesium chloride, 32p
MdCTP, 5mM DTT, 0.01% Triton X-1
00,50dg/ml actomyosin D, 50rnM
potassium chloride, 10 μw/ml oligo(dT), 2
0-50ug/ml mRNA portion, 10-60U/m
l reverse transcriptase (IUBCat, No, E, C.

After 90 minutes of incubation at 37° C., the reaction was terminated by adjusting the solution to 0.2% SDS, 20 mM EDTA. The solution was prepared in equal volumes 1:1 (
v/v) Phenol: Precipitated with chloroform and 2 volumes of absolute ethanol at -20°C and extracted with the concentrated liquid layer. The recovered cDNA was dissolved in water, adjusted to 0.1M with sodium hydroxide, and incubated at 60°C for 30 minutes.

After neutralization with acetic acid, the sample was treated with 50 mM sodium chloride and 0.
．． Chromatography was performed on a Sephadex G-150 column with a buffer containing 118D8. The labeled sample eluting into the empty column was collected and precipitated with 2 volumes of ethanol.

The size of @DNA was determined by electrophoresis in a 5% acrylamide gel using a buffer containing 90mM Trim, 90mM boric acid, and 2.5mM EDTA (pH 8,3) using a standard DNA fragment as a control. eD labeled with P
The location of NA was determined by gel autoradiography using Fuji-RX film. eDNA with more than 800 bases was cut and treated with 0.5M ammonium acetate, 9.01M magnesium acetate, 0.1% SDS, 0.5M ammonium acetate, 9.01M magnesium acetate, 0.1% SDS, 0.1%
It was extracted from the gel by electroextraction into a buffer containing 1mM EDTA. This high molecular weight eDNA was used for the synthesis of double-stranded samples.

One drawback of this method of transcribing cDNA is that it produces eDNA from every species present in the isolated mRNA population, ie, the eDNA becomes heterogeneous in sequence. One way to solve this is to use sequence-specific primers instead of oligo(dT) 12-18.

Another disadvantage of using cDNA primed with oligo(dT)12-18 is that transcription is
’ is stopped before reaching the point. This may be due to specific secondary structural features or generally less than optimal conditions for the enzymatic reaction. m
The absence of sequences derived from the 5-site of RNA is a significant drawback when expression of cloned sequences is the main goal. To specifically obtain 5' site sequences in eDNA, primers that hybridize with mRNA in the middle or near the 5' site can be used.

To do either of these, it is necessary to know the location of the amino acid sequence in the prochymosin protein, where there is a low degree of degeneracy in the genetic code. This means that the number of nucleotide sequences encoding a particular amino acid sequence is small enough to synthesize all possible combinations. We found that prochymosin has the following amino acid sequence.

The messenger RNA for this amino acid sequence would be as follows.

The central four codons of this clustered sequence have only fourfold degeneracy.A set of oligonucleotides complementary to a possible mRNA sequence can be expressed as follows.

One of these four sequences must exactly match the natural mRNA sequence and will therefore hybridize to the prochymosin mRNA site.

Other pairs of nucleotides for the same region are as follows.

These oligonucleotides can be synthesized using the conventional phosphotriester synthesis method (Shiung et al. (1979) NucA, A
c1d, R@1.61371-1385) and then an Ultrasil column (Alt@xL1mit
Purified by high pressure liquid chromatography (@dUSA). These synthetic oligonucleotides were used as primers for transcription of single-stranded eDNA from prochymosin mRNA.

2 μg of each oligonucleotide was purified using T4 polynucleotide kinase and 52P-ATP at the 5′ site.
Labeled with P phosphate.

The mixture contained 0.1-1.0 mC1 of P-ATP, 50
Contained: mM Tris, Cl (pH 7,8), 5 mM magnesium chloride, 10-β-mercaptoethanol, 5-20 units of T4 polynucleotide kinase. 37℃
After incubation for 30-60 minutes, the solution was diluted with an equal volume of 1;1 (v/
v) Phenol: Extracted with chloroform and the aqueous layer was extracted with 50-sodium chloride, 20mM Tris, Cl (pH
7.5) and passed through a Sephadex G-50 column equilibrated with 0.1% SDB. The removed portion containing the labeled oligonucleotide was allowed to accumulate and then precipitated by adding 2 volumes of absolute ethanol. The precipitate is 15.000×g
The relevant hearts were separated and collected for 10 min at 4°C.

The four labeled oligonucleotides are then converted to e as described above for oligo(aT)1z-1s, except when P-dCPT is not included in the reaction mixture.
It was used as a primer for DNA synthesis. Only one of the four oligonucleotides tested, i.e. 5'-GT
Only those with T, CAT, CAT, GTT-3' sequences were able to prime eDNA synthesis in the calf stomach mRNA population. This clearly indicates that the mRNA sequence in the region of the methionine doublet is complementary to the sequence of this particular oligonucleotide.

The cDNA produced had a maximum length of 650 bases as determined by gel electrophoresis, which was consistent with the predicted distance between the methionine-methionine sequence and the 5' site of the mRNA. However, it consisted of several types with different molecular weights. This eDNA is 1
It was a full chain and was labeled with one 52P-phosphate at the 5' site. These are Maxam and Silverado (Pro, N
att, Acad, Sc1. USA74560 (197
7) This is necessary for arranging DNA strands by the chemical method described in 9. The primed eDNA was sequenced by these methods to obtain a DNA sequence that matched the previously known amino acid sequence of the region of the polychymosin protein from which the primer nucleotide sequence was obtained. These sequencing methods are now commonplace in many laboratories, and M@thodsIngniymol
ogy68499-560 (1980) describes the step-by-step method.

The resulting sequence is shown in Figure 2 along with the relevant amino acid sequence and restriction enzyme recognition sites. Of particular interest are 8mml, Eeo, RI, HlndIII, BanM
The presence of a recognition site for the l enzyme.

Plasmid pAT153 lacks the Smm1 site used for initial cloning, and the other three sites are also rare. One or more of these may include specific 5'eD made from primer oligonucleotides.
Provides a useful site for binding NA to short eDNA made from oligo(dT) priming.

Ori is nucleotide 5'-GTT, CAT, CAT, T
The fact that the eDNA obtained using GG-1 is heterogeneous in size and can be used without fractionation in chemical sequencing reactions to give readable sequences makes it possible for eDNA to be This shows that a specific mRNA actually hybridizes to a specific point. This means that eDNA produced using oligonucleotides can be used as a fully specific hybridization probe in the screening of bacterial colonies obtained in cloning experiments (see screening procedures below). , this extended primer eDNA has a more stable interaction with plasmid DNA than the short oligonucleotide itself, making eDNA more suitable as a probe.

The transcription method described above produces a DNA molecule with a DNA "hairpin" structure at the 3' site. This can be used in making second strand DNA. Or 1
It can be used in two ways when making double-stranded DNA. Two methods can be used, single-stranded eDNA
For example, DNA polymerase 1 of Escherichia coli, or AMV, which under normal conditions would make a copy of single-stranded DNA, would make double-stranded eDNA in this case because the hairpin structure acts as a primer. It can be controlled by transcription enzymes.

In the preparation just described, single-stranded eDNA is
mMTrim, C1(p)(8,3), 20mMDTT
, 10mM magnesium chloride, 0.5mM dGTP, 0
．． 5mMdATP10.5mMdTTP, 0.5mMd
It was converted into double-stranded form by incubation in a mixture containing cTP, 2-10 μg/ml AMV reverse transcriptase. 4
After culturing at 5°C for 4 hours, the solution was diluted with 0.2% SDS, 20
Prepared in mMEDTA and then equal volume of 1:1 (v/v)
Phenol: Extracted with chloroform. The water layer is
Chromatography was performed on a Sephadex G-150 column as described above, and the extracted portions were pooled and precipitated with 2 volumes of absolute ethanol. Synthetic NA size company,
90mMTrls, 90mM boric acid, 2.5mM IDT
It was measured by gel electrophoresis using a standard DNA fragment as a control on a 5% acrylamide gel using a buffer containing As (pH 8,3).

The position of the labeled DNA was determined by gel autoradiography using Fuji-RX film.

Double-stranded eDNA with more than 800 bases is separated,
It was extracted from the gel in the same way as the single stranded version described above.

This high molecular weight version was used for cloning experiments.

The hairpin loop at one end of eDNA is α-ami.

(Bogt V'MEur, J, B.
loch@m33192-220 (1973))
It was carried out in a solution containing 25-50 units/ml of S1 and 25-50 units/ml of cDNA.

The “battered” residues at the ends of the double-stranded cDNA were removed by diluting the eDNA with 50mM Tris, Cl (pH 7).
, 5), 5mM MgCl2, 2mM mercaptoethanol, 0.0% each of the four deoxyribonucleotide phosphates.
5mM.

It is repaired by incubation with DNA polymerase II (Flenow fragment) in a 20 μl mixture containing 2 units of DNA polymerase I (so-called "Klenow fragment"). Incubation was for 15 minutes at 15°C. To ensure preservation of the nucleotide sequence at the ends of the eDNA gene (Rand et al. (1981) NueL, Ac1d.

An alternative method was used with some modifications to the operation of R@1.92251). Single-stranded eD prepared as described above
NA was 100mM sodium cacodylate (pH 7.1
), 0.1mM DTT, 10mM cold dCTP, 1mMC
oClA, 250 uCi/ml sH-dCTP, 10
For reactions containing ul/ml terminal transferase, 1
Oligodeoxycytosine was extended at the site. The reaction is
Measured by counting the incorporation of labeled dCTP and adding an average of 5G-100 residues of deoxycytosine to each molecule followed by termination with phenol extraction. Synthesis of the second strand to this elongated eDNA u, 10 sl/ml eDNA
A, 50mM TrLm, CL (pH 8,2), 30mM
KCtllomMMgCl2.50μl/- oligo d
This was carried out by annealing oligodeoxyguanosine (12-18 residues long) in a solution containing G(12-18). The mixture was incubated at 68°C for 30 minutes, then
10mM DTT 10.4mM each of the four deoxynucleoside triphosphates, containing 400 units/ml AMV reverse transcriptase
Chilled to ℃. The mixture was incubated for 10 minutes at 37°C, followed by 3 hours at 42°C, and then subjected to phenolic extraction and size fractionation as described above.

A more preferred method for introducing double-stranded cDNA into a plasmid is to use eDNA and a single tail of homopolymer oligonucleotide attached to the plasmid (
Otsuka (1981), Gsn@u339=346). DNA polymerase I (Klenow fragment) to trim the ends.
After incubation with eDNA, terminal transferase from calf thymus (EC2,7,7,3
1) was used to attach oligodeoxycytosine.

DNA of plasmid pA'r153 cut with Pat1
In A, then 10mM magnesium chloride and sH-dG
Oguanosine was attached using terminal transferase in the presence of TP. The reaction was measured by measuring the incorporation of the incorporation into the trichloroacetic acid precipitate, and after the incorporation indicated that there was an average of 30-40 residues attached to the 3' site The reaction has finished. The mixture was deproteinized using phenol and chloroform, and the tailed DNA was recovered by precipitation with an ethanolator.

Equimolar amounts of the two-tailed variety include 50st 0.15M Sodium Chloride, 0.01M Trim, U(
p) 17.4), 6 in a mixture containing 1mM EDTA
Annealed together by heating at 8° C. for 30 minutes, followed by cooling in a water bath to room temperature over approximately 4 hours. The annealed DNA was used to transform appropriate microbial cells as described below.

From the population of transformed organisms, selection was performed (perhaps based on sensitivity to tetracycline or ampsilin) to determine which organisms would successfully take up the recombinant DNA or not.

In this example, Katatsu et al. (1972J, Baat@rio
l.

Using the method of E. coli HBIOI (Boyer H
,W.

and Roland-Dousoix D, (1969) J, M6
1°8io1.41459-472) was transformed.

The cells are L- containing up to 100ug/mg ampicillin.
Agar culture dish (Miller J, ExperimentsinM
ol@cularGen@ticm.

Cold Spring Harbor Laboratory, New York, P433) and cultured at 37°C for 16 hours.

Clones were purified by streaking samples on L-agar plates supplemented with antibiotics and picking independent colonies. Recombinant clones (i.e., those resistant to tetracycline but sensitive to ampicillin) were tested in an initial "colony hybridization" screen using 52P-labeled mRNA fragments (Grunstein M.
, and Hogness D. (1975) Proe, Nat, Ae.
ad, Se1. (USA) 723961-3965). a) Crossbreeding of individual cloned DNAs
m from calf stomach in which a chymosin-specific sequence was found.
By comparing the RNA and b) mRNA from calf liver, which lacks chymosin-specific sequences, a number of potential chymosin-specific clones were selected. A more advanced colony hybridization screen using cDNA extended from the specific primers described above confirmed the nature of the positive chymosin clones.

DNA obtained from several such positive colonies was
Prochymosin mR by cross-priming (see above)
The aDNA obtained from NA showed a restriction enzyme degradation pattern that matched the known sequence. The DNA from these two clones was each more than half the length of the complete prochymosin gene. A portion of the DNA is the product of transcription from the 3' position of prochymosin mRNA to the 5' position of prochymosin mRNA, and the transcription was primed with oligo(dT) 12-18. The other part of the DNA is clearly polychymosin mRNA.
It was obtained by transcription from the 3' position of polychymosin mRNA to the 5' position of polychymosin mRNA. These DNA
These two clones containing fragments are 818 and 01 respectively.
A scale restriction site map of DNA from clones B18 and Gl is shown in FIG. 3(1). This figure shows the many restriction sites present in the overlap of DNA from G1 and 818 that join the two partial prochymosin genes to create the complete gene. Figure 3 3 (a
) and 3(b), the restriction sites are indicated as follows.

Pat=P, BamHI=B, KpnI=K, Ava■
=Av,SmaIwS,A1uIvmA%RI=R,B
A recombinant clone called g+11pm118 (Figure 3b) was created by joining the 3' part of clone A36 and the 5' part of clone Gl through a common RI site using common restriction and ligation methods. It was constructed by

Marxam and Gilbert technique (Proc, Nat, A
ead, Scl, U8A74560 (1977))
It has been used to determine the complete nucleotide sequence of clone 118. In Figure 3, the arrow is DN
The location and direction in which the arrangement of A is performed are shown. Dashed arrows are sequences obtained by extension of synthetic primers.

The results of this analysis are given in Figure 4, which shows the sequence of the coding strand and the corresponding amino acid sequence.

In general, this nucleotide sequence is consistent with previously published amino acid sequences for this protein. However, in this published sequence, amino acids 202 and 214 (indicated by asterisks in Figure 4) are aspartic acid. We showed that these amino acids are actually asparagine. Common chemical techniques for sequencing amino acids often mistake asparagine for aspartic acid, but nucleotide sequencing removed this confusion. We also found a short nucleotide sequence that codes for an amino-terminal adduct to the prochymosin protein. This suggests that prochymosin was originally created as preprochymosin. Although such a precursor of prochymosin has not been previously reported, it is likely that a variety of other secreted proteins (Davis B. , D., and Tye P. C. (1980
) similar to the known backbone found for Natur@283433-438).

The complete prochymosin gene (or other DNA construct obtained therefrom) isolated from a recombinant clone can be transferred into a vector designed to fully express the relevant protein.

The translation of a structural gene into a protein requires, among other things, the presence of a starting codon (ATC), which codes for the first amino acid of the protein to be expressed. Expression of preprochymosin occurs immediately since it starts from the methionine residue that is ATG. Expression of chymosin and prochymosin cannot occur subsequently. Instead,
A fusion protein must be expressed. The N-terminal appendage protein consists of a small protein with a methionine group as its first amino acid.

In the following example, the ATG codon is attached to chymosin or prochymosin, and the resulting expressed protein is
Methionine-chymosin (met-ehymos)
in) or methionine-prochymosine (mst-pro
chymoain). We prepared DNA constructs to express met-chymosin, met-prochymosin or preprochymosin in E. coli and also in yeast (Saccharomyces cerevisiae).

A, Structure of expression cilasmid in Escherichia coli 1, Methionine-prochymosin expression plasmid A36 (Fig. 3) was digested under standard conditions using the enzyme Pat■, and 900 bases were cloned containing the central part of the prochymosin gene. I inserted a pair. DNA
is significantly degraded in a very short time (30 seconds) using the enzyme Bal31, and the resulting molecule with non-binding ends and the synthetic H1ndm binding molecule (C, C, A,
A, G, C, T, T, G, G,) were ligated to them under standard conditions using T4 DNA ligase. This material was resolved with HlndII and then separated on a 5% acrylamide gel. A 900 bp fragment with Hlttd'm binding ends was isolated from the glue by disruption and soaking in buffer, then extracted with phenol, and then precipitated with ethanol. Then, Hl
It was recloned into pAT153 cut with nd■ and dephosphorylated with microbial alkaline phosphatase. This new plasmid was named C5.

When C5 DNA is digested with Hlnd■, dephosphorylated with alkaline phosphatase, and digested with RI, R1
A fragment (fragment I) having a 5'-phosphate in the binding site and a 5'-hydroxyl group in the HINDIII binding site was generated.

Plasmid B51 (Figure 3) was cut with BamII, and two chemically synthesized oligonucleotides were inserted into the binding site, one with a 5'-hydroxyl end and the other with a 5'-phosphate. Those with ends were joined together. These were synthesized by the method previously presented for the synthesis of synthetic cDNA primers.

This binding restores the sequence removed by BamHI digestion, which cuts at nucleotide 14 of the prochymosin gene (Figure 4). The combined DNA is RI
This produced a small fragment (fragment 2) containing the amino-terminal portion of polychymosin with a non-binding site having a 5'-hydroxyl group and an RI-binding site having a 5'-phosphate. , Fragment 1 (500 ng) and Fragment 2 (280 ng) were ligated using T4 DNA ligase, and the resulting product was dissolved in a 5% acrylamide gel.

The fragment corresponding to 1+2 was cut and extracted from the gel as described above.

This fragment was then cloned into a plasmid called pCTβ4 (see restriction map in Figure 5). This plasmid is obtained from pAT153 by cloning the E. coli trp promoter/operator fragment and phase T7 transcription termination fragment using techniques well known in the art. It involves the so-called “sheaine and delgano” (5hln
The nucleotide sequence of this plasmid around the start site is
It is as follows.

The restriction enzyme site between the SD sequence and ATO allows adjustment of the ATO-80 distance, which is an important factor in maximizing the expression rate. This plasmid also has a Bindl restriction site just 3' to the RI site. p
Cleavage of c'r54 with RI and subsequent digestion with 81 nuclease creates non-binding ends. 10g of DNA was mixed with 0.3M NaCl, 0.025M NmAe (pH 4,
6), digested with 300 units of Sl nuclease in a buffer containing 0.001 MZSO4 at 20°C for 30 minutes. Protein was removed from the solution using phenol, and DNA was recovered by precipitation with phenol.

Cleavage of the resulting non-binding molecule with SalI resulted in a short non-binding portion-SaAI fragment that was isolated from an acrylamide gel as described above. Further cleavage of pCT with Hlndl and Ball produced a large indl-Sal2 fragment, which was also isolated from acrylamide glue.

The large Hlnd■-8atE fragment (90ng), the small non-binding portion-SalI fragment (30ag), and the reconstituted prochymosin fragment (1+2) were ligated using T4 DNA ligase, and the resulting DNA was ligated using standard E. coli HB101 was transformed by operation. When eight prochymosin clones were isolated and sequenced around the ATG codon by the method described above, they were found to all have the same nucleotide sequence as follows.

The plasmid thus contains the coding sequence for the complete amino acid sequence of prochymosin with an additional methionine residue at the amino terminus. However, the distance between the SD and ATG sequences is too large to obtain good expression. This distance was reduced by cleaving the DNA at the Cla1 site and creating a non-binding variant by digestion with the 81 nuclease described above. This DNA is
Recombination with T4 DNA and transformation into E. coli HBIOI produced a plasmid called pCT70 (see restriction map in Figure 5). The plasmid DNA sequence around ATG is as follows.

The expression of met and prochymosin from pCT70 is as follows.

2. Expression of Methionine-Chymosin Construction of the chymosin expression vector began with the prochymosin plasmid pCT70 described above. pCT70DN
A was degraded using BeLl under conditions that DNA cleavage was not complete and full-length linear molecules were the main products. These cuts were made at both BclI sites in pCT70. Digestion of the DNA with R1, followed by fractionation and isolation of the largest fragments, yields a variety that extends from the R1 site around the met-prochymosin gene to the BeLI site closest to the starting point of the gene. It is now certain that it has grown. The small fragment needed to fully activate the met-chymosin gene moves the prochymosin gene into the R1 site (
Bam site at position 14 to y site) by insertion using a chemically synthesized binding agent such as
It was obtained from plasmid pCT57 (Fig. 5) obtained from pCT54.

This binding agent has 3 amino acids at the N-terminus that differ from the natural amino acid sequence.
(i.e., pseudo-prochymosin). pc'r57 plasmid DNA was restricted with Bat1,
A chemically synthesized binder molecule as follows was attached to the non-condensable BaL1 portion of the fragment using T4 DNA ligase.

The 5'-G, A, T, C portions of the new fragment are BamHl
Or it can be attached to a binding moiety from Bat1 or BgtI degradation. The DNA was further restricted using R1, resulting in the following fragments.

It contains the starting ATG and the nucleotides encoding the chymosin sequence up to the R1 site. This fragment was isolated from acrylamide glue. pc'r70 (90ng
) large R1-Bel\I fragment and small R1-GAT
When the C fragments (10 mg) were ligated together, a plasmid called pCT86, associated with BetI, was created. The DNA sequence in the SD to ATG region of this plasmid was determined by a general method as follows.

3. Expression of preprochymosin A plasmid called 118 (Figure 3) was degraded by Aval under standard conditions. From this K, - on the fragment
Nucleotides 14 to +2421 leave the 5' end of the preprochymosin sequence. The ends of this fragment are complementary to: chemically synthesized binder molecules (PG, T, C, T, G
,A.

T, C, A,) was bound to one end of this molecule with TA DNA, creating a fragment with the following structure.

This fragment was treated with DNA polymerase I to fill in the joining ends and then cut with Betl and Bgtl to provide 5'G, A, T, C, ends at both ends26.
A 0 bp fragment was generated.

pCT57 DNA was converted to BcLl under conditions of partial degradation.
The full-length linear molecule was then completely degraded with BgtI. The ATG from around only one BglH site
The large fragment extending to the Bel1 site near the was separated from the gel as described above. This large fragment (100 ng
) is a smaller 260 bp fragment (100 ng) K
When combined, a plasmid called pCT82 is created which contains all the sequences necessary to code for preprochymosin. The 260 bp fragment can bind to the large BeLa|BgLl [fragment from both directions; however, with the correct orientation, both Be1l and Bgt11 sites are regenerated, whereas with the wrong orientation, neither site is regenerated; As such, the correct orientation can be easily picked out by creating a constraint map. Although pCT82 was found to contain both of the renaturation sites, there are 28 bases from the SD to the ATC in the nucleotide structure around the start site. This cleaves the molecule at the BeLg site and then the B
It was reduced by removing a few bases with a brief digestion with eL31 nuclease.

Bidirectional cleavage of this enzyme produced unattached ends that were recombined to create variants with various values of SD to ATG distances.

B. Structure of yeast expression plasmids The five plasmids used for expression in yeast (Saccharomyces cerevisiae) are based on pBR322 and Q [mother 21 clone plasmids]. They are the subject of co-pending British Patent Application No. 8125934, and a partial restriction map of a common vector is shown in Figure 6. The yeast phosphoglycerate kinase gene (PGK) is a 5' Given the sequence, the gene insertion is one BanII
It can be done locally. It is for this reason that the entire DNA structure of E. coli was created in such a way that the entire gene can be cut into a Betl-BeLl fragment, which has a 5'-G, A
, with T, C, termini.

met. Chymosin, mct. The prochymosin and preprochymosin genes are pCT86, pCT70 and pC
They are excised from T82 by Bel1 digestion and ligated to pMA278 or pMA230, respectively.

In these two plasmids, the D around the insertion site
The NA sequence is shown in FIG.

1. Expression protocol in E. coli The protocol for expression of various genes is the same as that of all cilasmid vectors used.

A fresh 10 ml culture of E. coli (EBlol or RV308) containing the plasmid under study was grown in L-broth (Miller, J. H. (1972) Exp.
er1ments in Molecular Genet
les, Cold Spring Bar Parapolatory, New York.

p, 433) Ampicillin was supplemented to 100 ug/ml and cultured.

A 1:100 dilution of this culture is placed in M. medium (Miller, J. H. (1972)) supplemented with casamino acids, glucose, vitamin B, and ampicillin.

The "inhibition" medium contains 40-100 g/ml L-tryptophan, and the "induction" medium contains 10 g/ml of a specific tryptophan inducer - specific indoleacrylic acid added after 90 minutes of incubation. do. The culture was shaken at 37°C and the total incubation time was 3-4 hours. After this time, cells were collected by centrifugation, 10% (w/v) glycerol, 3% (w/v) S.D.
S, 6 containing 5% (w/v) mercaptoethanol
Dissolved by boiling in 0mM trin pH 6,8 buffer. Separated proteins were analyzed on a 12.5% acrylamide/SDS gel.

(Lasmmll UK (1979), NATURE22
7(680-685)e The gel was stained with Coomassie brilliant blue to visualize protein bands. In an experiment consisting of 35 newly synthesized proteins,
Labeled with 8-methionine. For this, the labeled amino acids were added after 3.5 hours of incubation to a final concentration of 20-30 uC/ml, and the incubation was continued for an additional 15-30 minutes until the cells were harvested and lysed. . Labeled proteins were detected on acrylamide gels and by autoradiography on Fuji-RX film.

2. Expression experiments The level of expression obtained from the recombinant plasmid and the level of the method used may be demonstrated by considering the case of plasmid pCT70 expressing methionine prochymosin, HB101 cells containing pc'r70. Grown under inducing conditions as described above, the synthesized proteins were examined on SO8/acrylamide gels. The cultures were grown in shake flasks (Figure 8) or fermentation vessels (Figure 9). Figure 8 shows a Trim-8DS gel of total E. coli proteins produced by pcT70 during 0-5 hour fermentation. Cells are M9 after 5 hours
The J” row grown to OD=i, 0 in the medium is a control containing 2 ug of authentic calf prochymosin. Trlm-SD of whole E. coli produced by
S gel is shown. Fermentation is controlled by dissolved oxygen 50% (110%)
Automatic foaming control was carried out at 37° C., 5 l volume and pH=7.2. OD6o after 9 hours.

=2.7. Row 'S'' is a control containing 2 μg of authentic calf prochymosin.

During both experiments, a protein band that corresponds to the position of authentic calf prochymosin is easily visible.

Proteins from cells containing pCT70 and cells containing POT54 (parental plasmid) are similarly 2
Original gel electrophoresis (P.H.Otsfarel (1975) J, Blol, Chem, 25040
07-4021). See Figure 10. 1st
In Figure 0, the arrowed point "A" indicates pCT70 and pC
The labeled protein spot common to both T54s is indicated. The point corresponding to EPC (prochymosin produced in E. coli by pCT70) is indicated by an arrow in Figure 10m. The dashed line marks the position of unlabeled authentic calf prochymosin added to the gel shown in Figure 10a.

Small cultures were labeled with 55B-methionine and total protein was analyzed as described. There was clearly visible extra protein in the cells carrying pCT70, which was shown to co-migrate with unlabeled calf prochymosin added extracellularly. From the gel analysis we estimate that approximately 50% of the total E. coli protein was expressed as methionine prochymosin in these cells. Product identification was further performed by antibody precipitation using polyclonal antisera prepared against authentic calf chymosin and prochymosin. 3
HB101/pCT7 labeled with 58-methionine
Total protein extracts from 0 were treated with these antisera and immunoprecipitated proteins were examined on an acrylamide/SDS gel. Figure 11 shows the results of this precipitation experiment. In the figure, the tracks on the gel are: a) Total pCT protein labeled with [''8] methionine; b) Total pCT protein labeled with [ss8] methionine precipitated with anti-prochymosine polyclonal antibody. Protein; c) Same as b) with extra real calf prochymosin (
d) unlabeled authentic calf prochymosin (AcP); d) unlabeled authentic calf prochymosin (AcP
C) movement position.

Excess protein in the pCT extract was selectively precipitated by both antisera, and the precipitate could be removed by adding excess authentic calf chymosin. Thus, this new protein is indistinguishable from authentic calf prochymosin with respect to its molecular weight, loading characteristics and immunologically determined sites.

3. Purification We devised a simple purification method to degrade methionine prochymosin from E. coli. Frozen E. coli/pCT70 cells grown under inducing conditions were grown at 0.05 times their weight.
Mtrls-HCjpH8, 1inMmTA.

It was suspended in 10% glycerin (v/v) containing 0.23 M NaCj, 130 ug/ml lysozyme, and the suspension was incubated at 4° C. for 20 minutes. Sodium dioxychlorate was added to a final concentration of 0.05% and 10 μg of DNAse X (bovine pancreas) was added per gram of E. coli starting material. The solution was incubated at 15°C for 30 minutes, over which time the viscosity of the solution decreased significantly e0.
01MtrlaHH81,0,1mM IDTA.

An equal volume of a solution containing 5% glycerin was added and the extract was centrifuged for 45 minutes at 10000 g at 4°C.

After further centrifugation as above, the supernatant was removed and the pellet was incubated at room temperature with 8M urea or 6M gucanidine hydrochloride.

0.05MHCtpH8, 1mM IDTA and 0.1M
It was rapidly dissolved in a buffer containing NaCj. Then 200 times the volume of 0.01 Mtrlm at 4°C overnight,
・HCLPH81”EDTAmo, IMNaCL and 1
Dialysis was performed against 0-glycerin. A heavy precipitate formed (insoluble in the E. coli protein) and was removed by centrifugation, leaving a solution of methionine prochymosin with a purity of approximately 75%. Figure 12 shows a Trim-SDS gel of various fractions during the initial steps of purification. The four tracks are: a) authentic calf prochymosin (AcPC); b) protein present in the cell pellet portion of E. coli pCT70; e) cell fragment pellet portion of prochymosin (EPC) produced by pCT70 in E. coli. Insoluble protein found in the fraction (after denaturation/reversion); d) Soluble protein found in the cell fragment pellet fraction of prochymosin (EPC) produced by pCT70 in E. coli protein.

Remaining impurities may be removed by chromatography on DEAE-cellulose or by gel filtration on Ultragel AeA54.

4. Activated prochymosin can be activated to active chymosin by exposure to acidic pH in the presence of E. coli extract. The extract was purified by adding 1M hydrochloric acid at 20°C.
The mixture of H2 and the mixture is stirred gently for 1 hour. A heavy precipitate forms which is removed by centrifugation at low speed. The clear supernatant is adjusted to pH 6 at 20° C. by adding 2M Trls pH 8. Bradford method (An
al, Blochsm.

(1976) 72248) shows that nucleic acid treatment removes 90% of the E. coli proteins in the extract.

In one experiment, 4 mg of genuine Shusei prochymosin
was mixed with 2.4 mg of clarified raw extract of E. coli K12. The mixture was acidified, centrifuged and neutralized. Next, add it to 0.025MNaP.

Ultroge equilibrated with 0.1M Na'CP pH6
It was applied to a 2.4 x 83 cm column of lACA54 at a rate of 15 ml/hr at 4°C. 2.51 portions were collected and tested for chymosin activity and absorption. Absorption at 260 nm and 280 nm was performed on each portion collected. The results of the absorption measurements are shown graphically in FIG. The graph shows a good separation of the chymosin moiety (marked) from the foreign moiety; the chymosin activity measurement (not shown) corresponds to the absorption peak for chymosin. The recovery of chymosin to coagulation activity was 40%.

5. Test procedure We have devised a rapid micromethod for testing the activity of chymosin, which involves incubation for a certain period of time in a microtiter plate. 12g of dry skim milk for 100g
6 ml of 20 mM calcium chloride at room temperature.
Leave for 0 minutes. 50 μt of sodium phosphate pH 6.3 buffer is placed in each well of the microtitration plate, 50 μt of each sample is placed in the first row of wells, and a series of dilutions is made across the plate. The microtiter plate containing the milk solution and the diluted sample was incubated at 37°C.
Incubate in the incubator; add 50 μt of milk solution to each well and return the plate to the incubator. After 15 minutes, the plate is removed and placed over the sink, allowing the liquid milk to run off, leaving a congealed depression.

The number of solidified wells in a series of dilutions indicates the amount of chymosin activity in the stock solution. Coagulate the solidified microtitration plate using a photocopier to keep a record of the test. This recording allows detection of 24 nanograms of chymosin in 15 minutes of incubation. Even lower amounts can be detected by increasing the incubation time. E. coli extracts do not coagulate milk or inhibit coagulation.

The test included EDTA (<5mM), DTT (<10mM)
- Glycerin (<12.5 chima/v), 'Tetler N-
Ptylammonium hydroxide ((0,2M)
It is not sensitive to most common buffer components containing . However, the young solvent, Twss
n80 (all concentrations), Nan1detNP-40 (
50% active at 0.08% v/v), sodium dioxychlorate (agile at 0.06% v/v), 5D
It is very sensitive to S (sensitive to 0.01 悌). The test has a salt optimum of 0.015M (NaCA) and is not sensitive to pH variations between 5 and 6.80.

In the above description, various embodiments of the invention are described with reference to the accompanying drawings.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the configurations of recombinant chymosin, prochymosin, gleprochymosin, and plasmid vectors. FIG. 2 shows the nucleotide sequence of aDNA obtained by transcription of mRNA using specific primers of synthetic oligonucleotides. FIG. 3 shows the restriction enzyme map of several clones obtained in (a), and the restriction enzyme map of clone 118 in (b). Figure 4 shows (prochymosin mRNA and chymosin mRNA)
The complete nucleotide sequence of preprochymosin mRNA (including the sequence of FIG. 5 shows a restriction map of plasmid vectors constructed for the expression of methionine-chymosin, methionine-prochymosin, and preprochymosin in E. coli. Figure 6 is a schematic diagram of the plasmid vector used for the expression of methionine-chymosin, methionine-prochymosin, and preprochymosin in yeast. Figure 7 is the DNA sequence around the BanII insertion site in the plasmid used for expression in yeast. This shows that. Figure 8 is after fermentation using a thin flask. Trlm-SDS gel chromatography of total E. coli proteins produced by pcT7G in HBIOI. Figure 9 shows the Trim-SDS of total E. coli proteins produced by pcT70 after fermentation in a 10L fermenter.
This shows gel chromatography. Figure 10 shows (a) isoelectric focusing/Tr of total E. coli protein labeled with 35S-methionine from pCT70.
is-SDS gel chromatography. Figure 11 shows the Trl of an experiment involving precipitation of prochymosin produced by pCT70 in E. coli by a polycloned antiserum of non-authentic calf prochymosin.
m-SDS gel chromatography. FIG. 12 shows Trim-SDS gel chromatography of various fractions during the initial stages of purification of prochymosin produced by pcT70 in E. coli. Figure 13 shows ultroggl AcA
Figure 3 shows the separation of genuine calf chymosin from substances present in an acidic crude extract of E. coli by gel permeation chromatography using 34. Agent Sho Ito abcd abed Continued from page 1 Priority claim [phase] November 11, 1981 ■ United Kingdom (
GB) ■8133998 ■December 1, 1981 ■United Kingdom (GB) ■8136185 ■February 10, 1982 Dispute United Kingdom (GB) ■p8203907 0 Inventor Michael Terence Dole United Kingdom of United Kingdom Patskinghamshire Studley Green Maycomb Road Utdbury (Street address) Inventor C. John Roy Harris, United Kingdom Berkshire, United Kingdom Bracknell Binfield, United Kingdom Berkshire 540 Inventor Peek Anthony Lowe, United Kingdom Berkshire, United Kingdom・Leading Melrose Avenue 9 [Phase] R. John Spencer Emtage, United Kingdom of United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, United Kingdom, 9th Avenue, Melrose Avenue, Benjamin House, 12 1 □: Procedural Amendment 1, ° 1 ν Showa 57 Patent Application No. 104672 2, Name of the invention Method for producing polypeptide 3, Person making the supplement 6, Number of inventions increasing IJI+ by drawing + E 7, Revised lunar diagram 1:... :' 8. Contents of amendment

Claims (1)

[Scope of Claims] 1. Methionine-chymosin, methionine-prochymosin or preprochymosin expressed by a host organism transformed with a vector system carrying a gene encoding methionine-chymosin, methionine-prochymosin or preprochymosin, respectively. A method for producing chymosin, which consists of an operation of cleaving the chymosin. 2. A method for producing chymosin consisting of the following operations. a) Insertion of the gene encoding methionine-prochymosin into the vector system. b) Transformation of a host organism with a gene having a vector system. c) Cleavage of methionine-prochymosin expressed by the host organism to produce chymosin. 3. A method for producing methionine-prochymosine comprising the following operations. a) Insertion of the gene encoding methionine-prochymosin into the vector system. b) Transformation of the host organism by the gene with vector system. c) Isolation of methionine-prochymosin expressed by the transformed host organism. 4. A method for producing preprochymosin comprising the following operations. a) Insertion of the gene encoding preprochymosin into the vector system. b) Transformation of the host organism by the gene with vector system. c) Cultivation of the host organism for expression. 5. The procedure of claim 4, wherein the preprochymosin expressed by the transformed host organism is isolated. 6. A method for producing methionine-chymosin comprising the following operations. a) Insertion of the gene encoding methioene-kimosi/ into the vector system. b) Transformation of the host organism by the gene with vector system. c) Isolation of methionine-chymosin expressed by the transformed host organism. 7. A method for producing prochymosin comprising the following operations. a) Insertion of the gene encoding preprochymosin into the vector system. b) Transformation of the host organism by the gene with vector system. c) Cleavage of preprochymosin expressed by the transformed host organism to produce prochymosin. 8. A method for producing chymosin comprising the following operations. a) Insertion of the gene encoding preprochymosin into the vector system. b) Transformation of the host organism by the gene with vector system. c) Cleavage of preprochymosin expressed by the transformed host organism to produce chymosin. 9. Chymosin produced by the method according to claim 1, 2 or 8. 10. Methionine-prochymosine. 11. Methionine-prochymosine produced by the method according to claim 1 or 3. 12. Preprochymosin. 13. Preprochymosin produced by the operation according to any one of claims 1, 4, and 5. 14. The gene encoding the aforementioned preprochymosin. 15. Gene encoding the aforementioned methionine-prochymosin. 16. Gene encoding the aforementioned methionine-chymosin. 17. A vector system containing the gene encoding methionine-prochymosin. 18. Vector system containing the gene encoding preprochymosin. 19. Vector system containing the gene encoding methionine-chymosin. 20. The vector system according to any one of claims 17 to 19, comprising a regulatable expression promoter sequence. 21. one or more microorganisms capable of transforming a host organism and imparting a phenotype to the transformed host organism that is different from the phenotype of the untransformed host organism; Claim 17 including the above arrangement
21. The vector system according to any one of Items 20 to 20. 22. The vector system according to claim 21, which comprises an E. coli tryptophan operon promoter sequence. 23, one or more capable of transforming a eukaryotic host organism and imparting a phenotype to the transformed host organism that is different from the phenotype of the untransformed host organism; 21. The vector system according to any one of claims 17 to 20, comprising more than one sequence. 24. The vector system according to claim 23, wherein the host organism is yeast. 25. The vector system according to claim 24 obtained from microbial plasmid pBR322 and yeast 2μ plasmid. 26. The vector system according to any one of claims 23 to 25, which comprises a yeast phosphoglycerate kinase (PGK) gene promoter sequence. 27. A host organism transformed by the vector system according to any one of claims 17 to 26. 28. A microorganism transformed by the vector system according to any one of claims 17 to 22. 2. Any one of claims 17 to 22
E. coli transformed by the vector system described in Section 1. 30. The E. coli strain according to claim 29, wherein the strain is RV308. 31. The E. coli strain according to claim 29, wherein the strain is RV308. 32, Claims 17, 18, 19,
A yeast transformed with the vector system according to any one of Items 20, 23, 24, 25, and 26. 33, Claims 17, 18, 19,
Saccharomyces cerepesiae (mouth eeharom.
ye aerv1mla@). 34, an oligonucleotide comprising the nucleotide sequence 5'GTT, CAT, CAT, GTT3', where G is guanine, T is thymine, A is adenine, and C is cytosine. 35. A method for detecting mRNA containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of chymosin, in which the origininucleotide of claim 34 is used as a hybridization probe. 36. A single-stranded eDNA encoding a polypeptide containing the amino acid sequence of chymosin, in which the origininucleotide of claim 34 is used as a transcription primer.
Method for preparing a portion of. 37. Assay for chymosin activity in which one or more dilutions of the sample are incubated with milk. 38. The method for assaying chymosin according to claim 33, which comprises the following operations. a) Preparation of serial dilutions of the sample. b) Addition of milk standard to each dilution. c) Incubation for a given time. d) Observation of whether the milk sample coagulates. 39. The assay method according to claim 37 or 38, wherein the milk is dry skim milk dissolved in water and reconstituted.